We are studying the mechanism whereby cultured cells develop resistance to Methotrexate as a result of increased synthesis of dihydrofolate reductase. We have shown in a number of mouse and hamster-derived cell lines that the increased synthesis results from the amplification of DNA sequences containing the dihydrofolate reductase coding sequences. We have also shown that in those cell lines with stably amplified genes, the dihydrofolate reductase genes are present on chromosomes, whereas in those cell lines with unstably amplified genes, the dihydrofolate reductase genes are present on extrachromosomal elements called "double minute chromosomes." Our future studies are designed to determine the mechanism of the gene amplification. We plan to study the early events in the development of resistance and to determine the mechanism whereby double minute chromosomes are generated, and how they replicate and whether they can integrate into chromosomes. Other studies involve characterization of RNA virus particles and nucleotide sequences in the resistant cell lines, and any possible role they may play in the amplification process. Other studies are directed at an understanding of the metabolism of dihydrofolate reductase mRNA, including studies on the processing of the mRNA precursor. Additional studies are directed at the mechanism whereby dihydrofolate reductase is synthesized at different rates in different parts of the cell cycle.